Understanding the cause of schizophrenia (Sz) is a major area of emphasis for NIMH. This R21 submission requests two years of support to develop a novel, widely applicable method to understand cortical circuitry abnormalities in Sz and other psychiatric/neurological disorders. The overall goals of this application are: (i) Establish a gene, cell-type and region-specific method to mimic molecular postmortem findings in Sz in an animal model;(ii) Use this method to test whether cell-specific knockdown of one such gene, GAD67(Gad1), causes gamma oscillation and cortical circuitry abnormalities typical of Sz. If successful, this project will: (i) Validate a novel method to investigate the function of genes implicated in disorders such as Sz involving cortical circuitry deficits;(ii) Generate a new animal model of Sz with good construct validity which can be used to develop new therapeutic approaches to improve gamma oscillations and cognition;and (iii) Determine whether downregulation of GAD67 expression can cause cortical circuitry deficits typical of Sz. In order to specifically knockdown (KD) gene expression we propose to use RNA interference (RNAi), building on our previous experience using this technique in the sleep field. We will initially target glutamic acid decarboxylase (GAD67), the major synthesizing enzyme for the inhibitory neurotransmitter GABA, since clinical postmortem data and animal models of Sz report a partial (30-50%) reduction in GAD67 levels in cortical interneurons expressing the calcium binding protein parvalbumin (PV). To achieve cell-type and region specificity, injections of viral vectors (adeno-associated virus;AAV) with Cre recombinase-dependent expression of small hairpin RNA targeting GAD67 (shRNA-GAD67) will be made in the prelimbic cortex of mice expressing Cre recombinase (Cre) in PV-positive neurons (PV-Cre mice). Thus, recombination and expression of shRNA will only occur in PV-Pos neurons in this cortical area, causing a cell and region specific KD of GAD67. The control vector will express scrambled shRNA not targeting any known mouse gene (shRNA- CTRL). The viral vectors will also express enhanced green fluorescent protein (GFP) in a Cre-dependent fashion as a marker of viral expression. Selectivity of viral expression will be determined by fluorescent immunohistochemical staining for PV and comparison of GFP/PV expression i.e. whether GFP is only located in PV neurons. Effectiveness of the KD of the target gene (GAD67) will be assessed by: (i) Quantitative Polymerase chain reaction (PCR) performed on mRNA harvested from transfected PV-Pos neurons using laser capture microdissection (LCM);(ii) Immunohistochemical staining for GAD67 protein and luminance measures of staining intensity. Selectivity of the KD will be confirmed by LCM/PCR for GAD65 and PV as well as immunohistochemistry for PV. We will use RNAi targeting GAD67 and PV-Cre mice to test whether a reduction in GAD67 specifically in PV-Pos neurons is a key event causing cortical circuitry deficits leading to reduced in vitro gamma (30-80 Hz) oscillations due to altered inhibitory synaptic transmission. PUBLIC HEALTH RELEVANCE: Schizophrenia is a major mental illness with negative impact on the individual, their family and on healthcare costs. Cognitive deficits in this disease are thought to be due to defective cortical circuitry, reflected as abnormal gamma oscillations in electroencephalographic recordings. This application aims to establish a new, broadly applicable method to understand cortical gamma abnormalities typical of schizophrenia in an animal model.